Effects of insulin insufficiency on forelimb and tail ... - Development

. Embryol. exp. Morph. Vol. 30, 2, pp. 427-447, 1973 427
Printed in Great Britain
<strong>Effects</strong> <strong>of</strong> <strong>insulin</strong> <strong>insufficiency</strong>
on forelimb and tail regeneration in adult
Diemictylus viridescens
BySWANI VETHAMANY-GLOBUS^ND
RICHARD A. LIYERSAGE
From the Ramsay Wright Zoological Laboratories,
University <strong>of</strong> Toronto
SUMMARY
Experimental diabetes caused by pancreatectomy resulted in a marked interference in limb
and tail regeneration in adult newts. Under pancreatic <strong>insufficiency</strong>, throughout the entire
period <strong>of</strong> regeneration, limb and tail regenerates (26 days old) consistently showed sparse
population <strong>of</strong> blastema cells, while older regenerates (58-79 days) exhibited severe abnormalities.
Newts with these abnormal regenerates always had an atrophied pancreas. In the event
that the cauterized pancreas regenerated, there was a subsequent recuperation in the regeneration
processes <strong>of</strong> the appendages. Our data strongly indicates that a correlation exists between
the normal (intact) pancreas and regeneration <strong>of</strong> the limb and tail in the adult newt.
In addition, alloxanization inhibited normal limb and tail regeneration. Again, recuperation
<strong>of</strong> the pancreas from the effects <strong>of</strong> alloxan was followed by restoration <strong>of</strong> limb and tail
regeneration. These results suggest an <strong>insulin</strong> role in the hormone control <strong>of</strong> regeneration.
The possible actions <strong>of</strong> <strong>insulin</strong> are discussed.
INTRODUCTION
Previous studies have demonstrated that the presence <strong>of</strong> an intact anterior
pituitary gland is essential in adult Diemictylus viridescens not only for the
survival <strong>of</strong> the animal but also for the normal regeneration <strong>of</strong> its appendages (see
reviews by Schotte (1961), Rose (1964), Schmidt (1968) and Thornton (1968)).
Although considerable interest has been directed toward the study <strong>of</strong> hormone
control <strong>of</strong> the regenerative processes in urodele appendages, the exact nature <strong>of</strong>
their influence is still not clear.
On the basis <strong>of</strong> a series <strong>of</strong> experiments, Schotte and his associates hypothesized
that the pituitary gland responds to the stress <strong>of</strong> limb amputation by releasing
ACTH which, in turn, promotes regeneration by stimulating the production
<strong>of</strong> the adrenocorticosteroid hormones (Schotte & Hall, 1952; Schotte &
Chamberlain, 1955; Schotte & Bierman, 1956; and Schotte & Christiansen,
1
Author's address: c/o Dr M. Globus, Department <strong>of</strong> Biology, University <strong>of</strong> Waterloo,
Waterloo, Ontario, Canada.
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428 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE
1957). They also postulated that the pituitary-adrenal synergism is probably
active in: (a) preventing dermal invasion under the wound epidermis, and
(b) supporting tissue dedifferentiation.
However, Wilkerson (1963) obtained limb regeneration when he injected
bovine somatotropin into hypophysectomized newts, beginning as late as 15 days
post-amputation. He concluded that the loss <strong>of</strong> regenerative ability in the adult
newt limb following hypophysectomy was due to the absence <strong>of</strong> growth hormone.
Several workers have shown that limb regeneration is thyroxine-dependent.
Following thyroidectomy normal limb regeneration is retarded but not completely
inhibited. Early removal <strong>of</strong> the thyroid gland interferes with dedifferentiation
and late removal interferes with the growth <strong>of</strong> the regenerate (Walter,
1910; Richardson, 1940; Schotte & Washburn, 1954). Antuitrin G (a commercially
prepared mammalian anterior pituitary extract) in combination with
thyroxine was more effective in promoting regeneration in hypophysectomized
adult newts than Antuitrin G alone (Richardson, 1945).
Recently, prolactin has been shown to have an effect on limb regeneration
(Connelly, Tassava & Thornton, 1968; Tassava, 1969). They showed that prolactin
alone increased survival and limb regeneration, but less effectively than
did prolactin in combination with thyroxine. From this evidence Tassava (1969)
postulated a 'prolactin-thyroxine' synergism operating in adult newt limb
regeneration.
All <strong>of</strong> these hormones that have been associated with regeneration in D.
viridescens, namely prolactin, cortisone, thyroxine and growth hormone, are
also known to induce the production <strong>of</strong> <strong>insulin</strong> from the islets <strong>of</strong> Langerhans
(Haist, 1959; Lazarus & Volk, 1962; Tepperman, 1965; Bern & Nicoll, 1968).
Prolonged treatment <strong>of</strong> mammals with these hormones can exhaust the islets
and, ultimately, cause diabetes; hence, these hormones are called 'diabetogenic
hormones' (review by Tepperman, 1965). In this connexion, Genuth & Lebovitz
(1965) observed that addition <strong>of</strong> ACTH to culture medium induced the production
<strong>of</strong> <strong>insulin</strong> in the islets <strong>of</strong> mouse pancreas in vitro. In consideration <strong>of</strong> these
findings, the hormones thus far related to limb regeneration in the adult newt
may conceivably act by stimulating the production <strong>of</strong> <strong>insulin</strong>. The following
experiments have therefore been designed to try to determine whether or not
<strong>insulin</strong> is involved in the hormone control <strong>of</strong> regeneration.
MATERIALS AND METHODS
Adult newts, Diemictylus viridescens used in this investigation, were obtained
from central Massachusetts, U.S.A. Medium sized animals <strong>of</strong> both sexes,
weighing approximately 1-8 g, were used. Prior to experimentation the animals
were allowed a 2-week acclimatization period and were kept in dechlorinated
tap water at 20 ± 1 °C and fed lean ground beef twice weekly.

Insulin and regeneration 429
Pancreatectomy
Adult newts, previously deprived <strong>of</strong> food for 3 days, were anaesthetized in
M.S. 222 (1 g: 1000 ml aqueous solution, Sandoz), prior to pancreatectomy. An
oblique incision, about 3 mm in length, was made in the left ventro-lateral region
<strong>of</strong> the abdomen in order to expose the pancreas. This diffuse gland lies between
the duodenum and the stomach and is enclosed in the hepato-gastric and dorsal
ligaments.
Pancreatectomy was achieved by cauterizing the gland with a Radiotom
(Siemens-Model 614) electro-cautery unit. The pancreas was cauterized until
the tissue became opaque; great care was exercised to avoid extensive damage
to the blood vessels supplying the gut and the spleen.
Following pancreatectomy, the operated area was rinsed with sterile amphibian
Ringer's solution, and the organs which were previously exteriorized
were returned to the body cavity; the incision was sutured using no. 6-0 Ethicon
eye sutures. Both sham pancreatectomized and control animals served to compare
the effects <strong>of</strong> surgery. All animals were allowed to recuperate for 2 days
post-operatively at 15 °C and then returned to 20 ± 1 °C. Their regular feeding
schedule was not resumed until 15 days post-pancreatectom.y. The water level
was maintained at a depth <strong>of</strong> about 0-5 cm which was sufficient to keep the
animals' skin moist until the incision had healed.
Alloxan treatment
Following the method <strong>of</strong> Falkmer (1961), alloxan was administered to adult
newts in an attempt to bring about beta cell destruction and ultimately induce
a state <strong>of</strong> <strong>insulin</strong> <strong>insufficiency</strong>. Adult newts, <strong>of</strong> uniform size (weighing approximately
2 g), were deprived <strong>of</strong> food for 3 days prior to the injection <strong>of</strong> alloxan
monohydrate (a 5 % solution, freshly prepared in citrate-phosphate buffer at
10 °C and pH 4-0). One group <strong>of</strong> animals received two intramuscular injections
<strong>of</strong> alloxan (each 0-3 mg per g <strong>of</strong> b.w.) into the midback region with a 2-day
interval between injections. The second group <strong>of</strong> animals received two injections
<strong>of</strong> 0-2 mg acid alloxan per g b.w. at intervals <strong>of</strong> 27 days between injections. The
controls received an equal volume <strong>of</strong> buffer solution. The longer interval between
injections in the second group was chosen in an attempt to minimize the
possible toxic effects <strong>of</strong> alloxan on the newt. All animals were kept at 10 °C,
for 24 h prior to injection and then for 2 additional days following the administration
<strong>of</strong> alloxan, whereupon they were returned to 20+1 °C (see Falkmer,
1961). Urine-sugar analyses were performed on all animals commencing on the
fourth day following alloxan treatment.
Daily urine-sugar analyses were made on pancreatectomized, alloxanized and
control animals, using Testape (Lilly and Co. Canada Ltd., Toronto) as a
method <strong>of</strong> indicating the amount <strong>of</strong> glucose (range = 0-1-2 %) in the urine.
Thus, the onset and duration <strong>of</strong> the diabetic state <strong>of</strong> the experimental animals
28-2

430 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE
was detected and recorded. The right forelimbs and tails <strong>of</strong> the pancreatectomized
animals were amputated at the onset <strong>of</strong> diabetes.
Upon termination <strong>of</strong> the experiments limb and tail regenerates and the pancreatic
tissues <strong>of</strong> the experimental and control animals were fixed in Bouin's
fluid. Limb and tail regenerates were decalcified in Jenkins' solution, sectioned
at 8 /an and stained with hematoxylin and counterstained with orange G-eosin.
Pancreatic tissue was stained with aldehyde fuchsin, Ponceau de xylidine-acid
fuchsin and counterstained with fast green (Epple, 1967).
RESULTS
<strong>Effects</strong> <strong>of</strong> pancreatectomy on forelimb and tail regeneration
Pancreatectomized animals excreting 0-1 % glucose in the urine survived for
periods <strong>of</strong> up to 52 days, but those excreting 0-25 % glucose died within 15 days
after pancreatectomy. It was difficult to keep animals alive in a prolonged state
<strong>of</strong> diabetes; nevertheless, a minimum period <strong>of</strong> 25 days from the time <strong>of</strong> amputation
is required to observe changes in limb regeneration. Therefore, the majority
<strong>of</strong> cases were fixed 25 days post-pancreactectomy. The results are summarized in
Table 1. The regenerates will be considered in two groups: Group A is composed
<strong>of</strong> regenerates fixed 25-35 days post-pancreatectomy; whereas Group B consists
<strong>of</strong> regenerates fixed 58-79 days after pancreatectomy.
Complete pancreatectomy in the newt resulted in death about 4 days after
surgery (Table 1, Group A, Series I). Cauterization <strong>of</strong> approximately 75 % <strong>of</strong>
the pancreas did not result in an observable difference between the control regenerates
and those <strong>of</strong> depancreatized animals, and also glucose was not detected
in the urine during the experiment (see Table 1, Group A, Series II). However,
when approximately 90 % <strong>of</strong> the pancreas was cauterized, inhibition <strong>of</strong> normal
limb and tail regeneration was observed (Series III, IV and V).
Limb regenerates
Group A: Series III and IV, Table 1, 25-28 days. By 25 days post-amputation,
the control regenerates showed advanced cone-shaped blastemata (Fig. 1). Bone
dedifferentiation had occurred and a dense population <strong>of</strong> blastema cells had
accumulated distal to the stump bone. Mitoses were frequently observed and
procartilage condensation was apparent in the blastema region (see reviews by
Schotte (1961), Rose (1964), Schmidt (1968) and Thornton (1968)).
A total <strong>of</strong> 110 adult animals (controls = 50; experimentals = 60) was included
in series III <strong>of</strong> which 31 experimental animals survived. Limb and tail regenerates
from these experimental animals were fixed 25-28 days following pancreatectomy.
Histological examination <strong>of</strong> the regenerates from these pancreatectomized
animals showed 11/31 cases with bone protrusion through the wound epidermis,
5/31 cases with epidermal blisters and 28/31 cases exhibiting sparse populations
<strong>of</strong> cells in the regeneration area. The remaining three cases showed near normal

Table 1. <strong>Effects</strong> <strong>of</strong> pancreatectomy on forelimb and tail regeneration in adult newt at 20 °C
Total
no. <strong>of</strong>
Total no. <strong>of</strong> Survival at end days
animals <strong>of</strong> expt. <strong>of</strong> re-
,
Group
K s / K Limb regenerates Tail regenerates
Control Control Experimental
^ genera-
Experimental
Con- Con- tion at
Abnor-
Nor- De- Nor- De-
and series trols Exptls trols Exptls fixation Normal mal Normal Abnormal Delayed mal layed mal layed
Complete pancreatectomy followed by amputation
— None recovered 20 — None fixed
Partial pancreatectomy (75 %) followed by amputation
— 12 = D3 — — 5 — 1 2 —
— 10 = CO — — 2 0 — 1 0 —
Partial pancreatectomy (90 %) followed by amputation
— 3 = CO 12 = Bp 10 = RC 48 — 3 28
20
4
0
20
40
20
D3
CO
5 =
20 =
35
25
12
10
5
20
20
10
5
20
Gr. A. Ser. I
Ser. II
(fl)
(b)
25-28 48 = CO
31
48
50 60
6 = EB
Partial pancreatectomy (90 %) 4 days post amputation
_ 3 = s 9 = RC 19 — — 14
2 = Bp
Partial pancreatectomy (90 %) followed by amputation
8 = D4; 17 = PAT 6 = CO 20 — 8 = PNR17 = RC;
PNR 9 = Dab AF;PAT
2 = S
Ser. Ill
3
26 19 = CO —
14
19
20 30
Ser. IV
20 40 20 25 58-79 20 = D4 —
Gr. B. Ser. V
Abbreviations (included in Table 1): AF = abnormal fin; Bp = bone protrusion; CO = cone stage; D3 = early three-digit regenerate; D4 = fourdigit
regenerate; Dab = digital abnormalities; EB = epidermal blister; PAT = atrophied pancreas; PNR = pancreas regeneration; RC = sparse
blastema cell population; S = stump (no regeneration).

432 SWANI VETHAMANY-GLOBUS AND R. A. LIYERSAGE
FIGURES 1, 2. The arrows indicate the level <strong>of</strong> amputation
Fig. 1. A longitudinal section through a 26-day forelimb regenerate <strong>of</strong> a control
adult newt showing a normal advanced cone-shaped blastema: b, blastema cells,
e, epidermis.
Fig. 2. A similar section through an experimental 26-day forelimb regenerate <strong>of</strong>
a partially pancreatectomized adult newt. The blastema cells (b) are sparse; growth
is inhibited as compared to the control. The stump bone lies close to the epidermis.
Magnification about x 75.

Insulin and regeneration 433
cone regenerates when fixed at 28 days post-amputation. These latter three
cases excreted sugar for the first 10-15 days post-amputation, and then stopped.
Neither bone protrusion nor epidermal blisters were observed in the control
animals. Procartilage cell condensation was not detected in any <strong>of</strong> the experimental
cases, indicating a delay in the rate <strong>of</strong> regeneration (Fig. 2).
In series IV partial pancreatectomy (90 %) was performed 4 days following
amputation on 30 animals; by this time the epidermis had completely covered the
amputation surface. Out <strong>of</strong> the 14 animals that survived for 26 days, two cases
had regenerates in which the humerus protruded through the epidermis; three
cases exhibited stumping (no regeneration); and nine showed markedly reduced
blastema cell populations compared to the controls.
Group A: reamputation experiments. Reamputation <strong>of</strong> 10 pancreatectomized
animals from series III was performed and subsequent regeneration over the
additional 25 days was compared with the original 25-day regenerates. The
diabetic state <strong>of</strong> these animals was monitored by daily urine-sugar analyses, as
previously described. Although the survival rate among these re-amputees
amounted to only two cases, interesting results were obtained. The first and
second limb regenerates from one diabetic animal were similar in their morphological
and histological appearance and in their rate <strong>of</strong> regeneration. That is to
say, both regenerates exhibited reduced blastema cell populations compared to
control regenerates and the diabetic state persisted throughout the experiment
(total <strong>of</strong> 50 days). However, the second regenerate <strong>of</strong> the other case showed
a considerably advanced limb morphogenesis over the first regenerate. Interestingly
enough this was preceded by the recovery <strong>of</strong> the animal from diabetes.
This animal excreted sugar in the urine for 35 days and thereafter the urine was
free <strong>of</strong> sugar. The first regenerate showed a sparse population <strong>of</strong> cells and resembled
the delayed regenerate <strong>of</strong> the diabetic animals previously described;
cartilage condensation was not apparent. This was in sharp contrast to the
second regenerate <strong>of</strong> this case which showed cartilage differentiation, dense
blastema cell aggregation and numerous mitotic figures. (Figs. 3-5). Restoration
<strong>of</strong> normal limb regeneration in the second regenerate was correlated with
a correction in the diabetic condition <strong>of</strong> the animal, as evidenced by the sugarfree
urine and the regenerated pancreas.
Group B: series V {Table T), 58- to 79-day regenerates. Control limb regenerates
from all 20 newts exhibited four, well-formed cartilaginous digits by 79 days
post-amputation and the morphological pattern <strong>of</strong> the regenerates resembled
that <strong>of</strong> normal forelimbs.
Twenty-five pancreatectomized animals that stopped excreting sugar within
10 days post-pancreatectomy were allowed to regenerate for 58-79 days. Out
<strong>of</strong> the 25 experimental animals, eight regenerated normal limbs and tails. These
eight animals appeared healthy and accepted food readily. An examination <strong>of</strong>
their viscera revealed a partial regeneration <strong>of</strong> the pancreas. The histological
preparations showed the presence <strong>of</strong> normal acinar and islet tissues.

434 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE

Insulin and regeneration 435
The remaining 17 pancreatectomized animals, however, exhibited either
abnormal or no regenerates. These animals refused food, lost weight and became
extremely thin. Limb regeneration was totally absent in two cases, even after
58 days post-amputation and abnormal in nine others. Of these nine cases four
exhibited spike-like regenerates, whereas five showed a lack <strong>of</strong> digital components
and considerable retardation <strong>of</strong> regeneration. Histological examination <strong>of</strong> these
four spike-like regenerates revealed complete absence <strong>of</strong> the radius and the
regenerates had only one digit giving a spike-like form to the new limb (see
Figs. 6, 7). Also, muscle and connective tissue development was considerably
reduced. The five cases that exhibited a lack <strong>of</strong> digital components were short,
thin and stunted. Histologically they showed one digital cartilage condensation
in only one <strong>of</strong> the five regenerates; the others were digitless. In addition to the
above-mentioned abnormal cases, extremely delayed regeneration was observed
in three cases exhibiting only a cone stage at 60 days. The remaining three
animals showed no signs <strong>of</strong> limb regeneration up to 32 days post-amputation
whereupon small cone-like limb regenerates began to appear, 58 days after
amputation.
The cauterized pancreas <strong>of</strong> the animals that exhibited abnormal limb and
tail regeneration was considerably reduced in bulk and severe atrophy was
observed in the acinar as well as in the islet tissue. The nuclei were pycnotic
and surrounded by only scanty amounts <strong>of</strong> cytoplasm; the latter was significantly
lacking in zymogen granules.
Tail regenerates
Group A: Series III and IV {Table 1), 25- to 28-day tail regenerates. The control
regenerates were approximately 2-2 mm long by 25 days post-amputation, and
showed well-differentiated dorsal and ventral fins, spinal cord and muscles.
Maturing cartilage and vertebral segmentation were evident. A comparison
between the control and experimental tail regenerates revealed considerable
delay in the rate <strong>of</strong> regeneration in the pancreatectomized animals. Mitotic
figures were infrequent in the sparse populations <strong>of</strong> blastema cells.
FIGURES 3-8. The arrows indicate the level <strong>of</strong> amputation
Fig. 3. A longitudinal section through a 25-day forelimb regenerate <strong>of</strong> a control
adult newt, showing a normal advanced cone stage regenerate; procartilage condensation
(c) is initiated. Magnification about x 90.
Fig. 4. A longitudinal section through a 25-day forelimb regenerate <strong>of</strong> a partially
pancreatectomized newt showing a delay in regeneration as compared to the control
(Fig. 3). Sugar excretion was observed throughout the entire period (25 days) <strong>of</strong>
regeneration, b, blastema. Magnification about x 90.
Fig. 5. A similar section through a 25-day forelimb regenerate from the reamputee
<strong>of</strong> the same animal (Fig. 4). The animal stopped excreting sugar after the first 10 days.
Note the palette stage with advanced cartilage differentiation (c). Magnification
about x 80.

436 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE

Insulin and regeneration 437
Group B: Series V, 58- to 79-day tail regenerates. The control tail regenerates
showed advanced growth and differentiation by 79 days post-amputation, and
exhibited well-defined vertebral arches (4 to 7 in number). Also spinal cord,
sympathetic and spinal ganglia, skin glands, blood vessels, tail musculature
and fins were well-differentiated at this time. In contrast, regeneration in the
17 experimental animals was extremely delayed, as evidenced by the small,
retarded regenerates (Figs. 8, 9). Moreover, growth <strong>of</strong> the fins was delayed in
relation to the central axis <strong>of</strong> cartilage and spinal cord; this resulted in a spikelike
appearance. (This type <strong>of</strong> regeneration was also observed in hypophysectomized
newts by Vethamany, 1970.) Mitoses were less frequently seen and
some rudimentary cartilage differentiation was evident. Tail regeneration in the
remaining eight experimental cases, however, was quite normal as indicated in
Table 1 and as similar to Fig. 8.
<strong>Effects</strong> <strong>of</strong>alloxan treatment
Twenty animals received two injections <strong>of</strong> 0-3 mg <strong>of</strong> alloxan per g <strong>of</strong> b.w.
at 2-day intervals. The right forelimbs and tails were amputated 2 days following
the second injection. However, four animals survived up to 55 days. The amputated
forelimbs and tails <strong>of</strong> these four animals showed abortive regenerates;
that is to say, they had permanent blastemata showing sparse populations <strong>of</strong>
cells. The distal tip <strong>of</strong> the stump bone was enveloped by a collar <strong>of</strong> cartilagenous
tissue and osteoclastic erosion <strong>of</strong> the bone was still much in evidence at 55 days
post-amputation. Mitotic activity was minimal, extensive vacuolization was
noticeable in the cells, and there was no trace <strong>of</strong> cartilage differentiation even
up to 55 days following amputation. However, the control animals (10), on the
other hand, exhibited well-formed forelimb regenerates by this time and showed
normal regrowth <strong>of</strong> the humerus, radio-ulna and four digits and had welldeveloped
limb musculature (Figs. 10, 11).
In the second set <strong>of</strong> animals (20 = experimentals and 10 = control) the
Fig. 6. A longitudinal section through a 62-day forelimb regenerate <strong>of</strong> a control
animal. Note the well-formed cartilaginous limb skeleton and musculature. Magnification
about x 25.
Fig. 7. A similar section through a 62-day forelimb regenerate <strong>of</strong> a partially pancreatectomized
adult newt showing an abnormal spike-like regenerate. Note the absence
<strong>of</strong> radius and many digital components. Only one digit (d) is seen. Muscles and
connective tissue in the regenerate are considerably reduced as compared to the
control. Magnification about x 25.
Fig. 8. A mid-sagittal section <strong>of</strong> a 62-day tail regenerate <strong>of</strong> a control newt showing
well-differentiated regenerate with six vertebral arches (v), spinal cord (s) differentiation
<strong>of</strong> centra in the cartilage (c) and fins (/). Magnification about x 40.
Fig. 9. A similar section <strong>of</strong> a 62-day tail regenerate <strong>of</strong> a partially pancreatectomized
adult newt exhibiting extremely small and retarded regenerate as compared to the
control above. Magnification about x 50.

438 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE

Insulin and regeneration 439
experimental received two injections <strong>of</strong> 0-2 mg acid alloxan/g <strong>of</strong> b.w. at
intervals <strong>of</strong> 27 days between injections; the right forelimbs and tails were amputated
2 days following the second injection. Five <strong>of</strong> these alloxanized animals
survived for 47 days post-amputation. Urine-sugar excretion (0-1 %) continued
for 25 days following amputation and thereafter sugar was not detected in the
urine. The amputated limbs and tails did not exhibit external signs <strong>of</strong> regeneration
until 33 days post-amputation and they had the appearance <strong>of</strong> non-regenerating
stumps. However, small cone-like blastemata were seen breaking through the
stump region by 47 days post-amputation (Figs. 12, 13). The forelimb and tail
regenerates as well as the pancreas <strong>of</strong> three animals were fixed at this time; the
remaining two cases were fixed 57 days post-amputation. The amputated limbs
and tails <strong>of</strong> the control cases regenerated quite normally, and by 47 days postamputation
they displayed normal advanced four-digit regenerates (see Fig. 12).
Histological examination <strong>of</strong> the three 47-day cone-shaped limb regenerates
from the second set <strong>of</strong> alloxanized animals revealed an accumulation <strong>of</strong> blastema
cells and pr<strong>of</strong>use vascularization <strong>of</strong> the region. Cell proliferation was active as
evidenced by the abundance <strong>of</strong> mitotic figures among the epidermal and blastema
cells. However, there was no detectable cartilage differentiation by 47 days postamputation.
Interestingly enough, the appearance <strong>of</strong> cone-shaped blastemata
and the sudden burst <strong>of</strong> mitotic activity was preceded by the disappearance <strong>of</strong>
glucose from the urine. The two limb regenerates which were fixed 57 days postamputation
were heteromorphic but exhibited some cartilage differentiation.
The tail regenerates from these alloxanized animals exhibited a delay in the
regeneration; the 47- and 57-day-old regenerates resembled controls <strong>of</strong> 22 days
<strong>of</strong> regeneration in their morphological and histological appearance.
The acinar cells <strong>of</strong> alloxanized pancreas (first set <strong>of</strong> animals) were packed with
dense zymogen granules that stained a deep purple with basic-fuchsin; this is
indicative <strong>of</strong> normally functioning exocrine tissue. However, compared to
FIGURES 10-13
Fig. 10. A longitudinal section <strong>of</strong> a 55-day forelimb regenerate from an adult newt
exhibiting normal regenerate with four digits. Magnification about x 30.
Fig. 11. A similar section <strong>of</strong> a 55-day forelimb regenerate <strong>of</strong> an alloxanized adult
newt exhibiting arrest in regeneration. Note the permanent blastema (b) and the
thick apical cap (e). Magnification about x 30.
Fig. 12. A longitudinal section through a 47-day forelimb regenerate <strong>of</strong> an alloxanized
adult newt exhibiting restoration <strong>of</strong> regeneration. Note the small coneblastema;
extremely delayed as compared to the control (Fig. 13). The pancreas <strong>of</strong>
this animal exhibited hypertrophy <strong>of</strong> the islets (see Fig. 16). Vacuoles (v) are seen near
the distal end <strong>of</strong> the stump bone. Note the accumulation <strong>of</strong> blastema cells (b).
Magnification about x 85.
Fig. 13. A longitudinal section through a 47-day forelimb regenerate <strong>of</strong> an adult newt
which served as a control for the alloxanized animal (Fig. 12). The regenerate
is well-differentiated with four digits. Magnification about x 30.

440 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE

Insulin and regeneration 441
controls only a few islets were ever observed in the alloxanized pancreas. Beta
granules were not easily discernible and vacuoles were seen in the islet cells
(compare Figs. 14 and 15). The pancreas from the second set <strong>of</strong> alloxanized
animals was conspicuously larger and more dense than those <strong>of</strong> the controls.
Cells <strong>of</strong> the exocrine part <strong>of</strong> the gland were normal and heavily packed with
zymogen granules. The most striking feature was the extensive hyperplasia
observed in the islets. The islets were conspicuously larger in size and were
found to extend the whole width <strong>of</strong> the gland (Fig. 16). This hyperplastic activity
<strong>of</strong> the islets, which was seen in the second set <strong>of</strong> alloxanized animals, suggests
that the islets had recuperated from the alloxan damage and the animals
recovered from alloxan diabetes (urine-sugar not detected after 25 days following
amputation).
DISCUSSION
In these experiments, the effects <strong>of</strong> <strong>insulin</strong> <strong>insufficiency</strong> are primarily manifested
in: (1) the failure <strong>of</strong> the blastema to accumulate a sufficient population <strong>of</strong>
cells in the regeneration area; (2) reduced cartilage formation in the limb and
tail regenerates; and (3) delayed growth and differentiation <strong>of</strong> the fins and
spinal cord in the tail regenerates.
Some <strong>of</strong> the partially depancreatized animals maintained normal health,
recovered from glucosuria, ate well and regenerated normal limbs and tails.
Paralleling these results was the fact that the pancreas regenerated in these
animals, whereas the cauterized pancreas <strong>of</strong> other animals exhibited extensive
atrophy <strong>of</strong> the entire gland. These latter animals lost weight gradually, ate poorly
and exhibited stunted, abnormally small regenerates. The factors responsible
for regeneration <strong>of</strong> the cauterized pancreas in some cases and atrophy in others
are not clear. Nevertheless, animals with an atrophied pancreas consistently
produced abnormal regenerates, whereas animals that regenerated the pancreas
also regenerated normal limbs and tails. This lends support to the possible
existence <strong>of</strong> a direct relationship between the functional state <strong>of</strong> the pancreas and
limb and tail regeneration in newts.
In addition to partial pancreatectomy, alloxanization was utilized as an
Fig. 14. A transverse section through the islet <strong>of</strong> an adult newt which served as
a control for the alloxanized animals. The islets lie between arrows. The beta cells (b)
are centrally located, easily seen with their prominent nuclei and granular cytoplasm.
This islet shows extensive vascular supply. Magnification about x 590.
Fig. 15. A transverse section through the pancreas <strong>of</strong> an alloxanized adult newt
(55 days post-alloxanization). This islet (/), seen between arrows, shows vacuolative
damage (v) and is not well defined as compared to the islet <strong>of</strong> the control newt. See
Fig. 14. The exocrine part (x) is seemingly unaffected. Magnification about x 590.
Fig. 16. A section through an alloxanized pancreas (47 days post-alloxanization)
showing regenerated islets. The islets have undergone hypertrophy. They are larger
compared to the control islets. Magnification about x 145.

442 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE
Adenohypophysis
Hydrocortisone Thyroxine Prolactin Growth
hormone
Regeneration
Pancreas I
Fig. 17. Hormone interactions in appendage regeneration in adult Diemictylus.
, Established relationships; , present investigation; , possible additional
pathways involving <strong>insulin</strong>.
additional approach to study the effects <strong>of</strong> <strong>insulin</strong> <strong>insufficiency</strong> on regeneration
processes. Alloxan selectively causes lesions in the beta cells, thereby leading to
diabetes mellitus in mammals. Lukens (1948) suggested that the action <strong>of</strong>
alloxan on the islets <strong>of</strong> the pancreas is direct and the resultant pathological
changes are the consequence <strong>of</strong> an immediate injury to the beta cells. In the
alloxanized newts, limb and tail regeneration was inhibited in most cases, and
the effects were more drastic than in the partially pancreatectomized cases.
However, some alloxanized newts showed recuperation <strong>of</strong> the pancreas from
alloxanization. This was followed by the subsequent restoration <strong>of</strong> limb
regeneration. The above evidence strongly indicates that a relationship exists
between the functioning pancreas (more specifically <strong>insulin</strong>) and the regeneration
<strong>of</strong> appendages in adult newts.
Corroborating these findings, are the recent in vitro experiments <strong>of</strong> Vethamany
(1970) which demonstrated that the presence <strong>of</strong> <strong>insulin</strong> is essential in promoting

Insulin and regeneration 443
growth and differentiation <strong>of</strong> the blastema in culture. This in vitro work further
indicates that the collective influence <strong>of</strong> the hormones (<strong>insulin</strong>, growth hormone,
hydrocortisone and thyroxine) is greater than the effect <strong>of</strong> any <strong>of</strong> them
individually.
In order to appreciate the role <strong>insulin</strong> might play and to gain more insight
into the hormone control <strong>of</strong> regeneration, the inter-relationships between <strong>insulin</strong>
and other hormones should be considered. Removal <strong>of</strong> a hormone from the
circulation produces widespread derangements involving the whole metabolism
<strong>of</strong> the animal which in turn will undoubtedly affect the production, the feedback
mechanisms and the action <strong>of</strong> other hormones. Thus, the interactions and
interdependence <strong>of</strong> hormones cannot be overemphasized. Figure 17 is a diagrammatic
representation <strong>of</strong> possible pathways <strong>of</strong> hormone interactions in
regeneration <strong>of</strong> appendages in adult newt. The dark, thick lines represent the
current investigation which provide evidence to link <strong>insulin</strong> to regeneration.
Previous work regarding the involvement <strong>of</strong> other hormones in regeneration are
depicted by thin unbroken lines, whereas the dotted lines represent the speculations
put forth by the authors (presently under investigation) as to the possible
pathways <strong>of</strong> hormone interaction in the regeneration process.
According to the hypothesis <strong>of</strong> Schotte (1961) and co-workers the pituitary
gland responds to the stress <strong>of</strong> amputation by releasing ACTH which, in turn,
stimulates the production <strong>of</strong> the adrenocorticoid hormones. In addition, they
suggest that the pituitary-adrenal synergism is probably active in the early
phases <strong>of</strong> limb regeneration by preventing dermal invasion <strong>of</strong> the subapical
space. They also propose that the pituitary-adrenal synergism is not active in
the later differentiative and morphogenetic phases <strong>of</strong> regeneration.
Although Schotte and his group stressed the importance <strong>of</strong> an anterior
pituitary-adrenal synergism in the regeneration processes <strong>of</strong> the limb, they have
not excluded other possibilities. To quote Schotte & Bierman (1956): 'in view
<strong>of</strong> our ignorance <strong>of</strong> the pathways, particularly in amphibia, <strong>of</strong> hormonal interactions,
one must remain hesitant in assuming as self-evident that the action <strong>of</strong>
ACTH in regeneration can be explained by its stimulatory role upon the
adrenals.'
It is known that corticosteroid-induced hyperglycemia causes morphological
changes in the pancreatic islets; this is indicative <strong>of</strong> increased <strong>insulin</strong> output
which restores blood sugar homeostasis in mammals (see Volk & Lazarus, 1963).
Wurster & Miller (1959) have also shown, in the salamander (Taricha torosa),
that hydrocortisone injections produce hyperglycemia and subsequent beta cell
degranulation. Although the results <strong>of</strong> the present investigation do not preclude
the possibility <strong>of</strong> a direct effect <strong>of</strong> ACTH-stimulated corticosteroids on regeneration,
it is equally possible that corticosteroids also promote regeneration indirectly
by stimulating the production <strong>of</strong> <strong>insulin</strong>.
In addition, ACTH has been shown to stimulate directly the release <strong>of</strong> <strong>insulin</strong>.
At this point, mention should be made <strong>of</strong> Schotte's experiments, in which he
2

444 SWANI VETHAMANY-GLOBUS AND R. A. LIVERSAGE
obtained better results, in terms <strong>of</strong> animal survival, the rate <strong>of</strong> regeneration and
the degree <strong>of</strong> morphogenesis, when he injected ACTH rather than cortisone
acetate into hypophysectomized animals. Thus, it is possible that ACTH promotes
limb regeneration in hypophysectomized animals by directly stimulating
the production <strong>of</strong> <strong>insulin</strong> in addition to its effect on the adrenals.
In addition to the corticosteroids, other hormones which have been shown to
support limb regeneration are also known to stimulate <strong>insulin</strong> production. For
example, growth hormone injected into hypophysectomized adult newts promotes
normal limb regeneration (Wilkerson, 1963). There are also data available
which show that limb regeneration is dependent upon the presence <strong>of</strong> an intact
thyroid gland (Schmidt, 1968; Schotte & Washburn, 1954). Connelly et ah
(1968) have shown that prolactin, in combination with thyroxine, promotes
limb regeneration in hypophysectomized animals.
There is an interesting correlation between the hormones mentioned above
and the production <strong>of</strong> <strong>insulin</strong>. Growth hormone, prolactin, thyroxine, corticosteroids
and ACTH are known to stimulate the production <strong>of</strong> <strong>insulin</strong> (Bern &
Nicoll, 1968; Haist, 1959; Lazarus et al 1962; Tepperman, 1965). It has also
been shown that hypophysectomy in addition to inhibiting limb and tail regeneration
in adult newts, results in the atrophy <strong>of</strong> the pancreas (Vethamany,
1970). Thus the possibility exists that all <strong>of</strong> these hormones promote limb and
tail regeneration either directly and/or indirectly by stimulating the production
<strong>of</strong> <strong>insulin</strong> (see Fig. 17). Since hypophysectomy or pancreatectomy brings about
widespread derangements in the metabolism, it is highly unlikely that only one
specific hormone is involved in regeneration. It is more likely that these hormones
do not act in isolated pathways but interact interdependently through
their effects on the metabolism.
When the source <strong>of</strong> <strong>insulin</strong> is removed from an animal, a marked sequence <strong>of</strong>
events occurs which is intricately interconnected. These events involve not only
carbohydrate metabolism, but also fat, protein and nucleic acid metabolism as
well as electrolyte and water balance (see review by Tepperman, 1965). Regeneration
processes in adult newts also involve carbohydrate, lipid, protein and
nucleic acid syntheses (see review by Schmidt, 1968). In response to severe
injury in mammals, large quantities <strong>of</strong> corticosteroids have been found in the
circulation during the first 24 to 48 h and, as a result, hyperglycemia ensues
(Schmidt, 1968). If this is true in urodeles, hyperglycemia is likely to stimulate
<strong>insulin</strong> production. Schmidt (1968) has reported that glycogen and lipids are
synthesized in the blastema cells prior to differentiation; these are probably
utilized, later, as a substrate reservoir for metabolic events essential to the cells
in regeneration. Insulin might play a role in this initial synthesis <strong>of</strong> glycogen
and lipids.
Chalkley (1959) has shown that mitotic activity during regeneration reaches
its maximum level around 25-31 days after amputation. Also studies concerning
the inhibition <strong>of</strong> mitotic activity, using X-irradiation (Butler, 1933) and colchi-

Insulin and regeneration 445
cine (Thornton, 1943) in regenerating systems, have shown that cell proliferation
is a prerequisite for normal regeneration to ensue. There is also evidence for
increased DNA-synthesizing activity <strong>of</strong> blastema cells during regeneration (Hay
& Fischman, 1961). Insulin <strong>insufficiency</strong>, in the current experiments, resulted in a
reduced blastema cell population and extremely retarded regeneration. Further,
the addition <strong>of</strong> <strong>insulin</strong> to cultures enhanced cell proliferation (Vethamany,
1970). Since the production <strong>of</strong> new blastema cells must surely involve DNA
synthesis one can invoke a DNA synthesis-stimulating role to <strong>insulin</strong>. Similar
enhancement <strong>of</strong> DNA synthesis was also reported in the alveolar epithelial cells
<strong>of</strong> mammary gland tissue in vitro when <strong>insulin</strong> was added to the medium
(Stockdale, Juergens & Topper, 1966).
Schmidt (1968) has shown, through quantitative estimations <strong>of</strong> total nitrogen
in regenerating forelimbs (D. viridescens), that there is active protein synthesis
during the initiation as well as the differentiation phases <strong>of</strong> regeneration. Burnet
& Liversage (1964) and Liversage & Colley (1965) obtained retarded and
abnormal limb regeneration when they used chloramphenicol and puromycin
to inhibit protein synthesis. Since <strong>insulin</strong> is known to increase the protein
synthetic capacity in diabetic rats (Wool et ah 1968), it is possible that <strong>insulin</strong> is
involved in protein synthesis during regeneration. Insulin is also known to
increase RNA synthesis (Steiner & King, 1966; Wool et al. 1968) and may well
exert an influence on RNA synthesis in limb regeneration. Future work along
these lines will elucidate the exact role played by <strong>insulin</strong> in the metabolic processes
involved in regeneration.
We wish to express our appreciation to Dr M. Globus for his interest and valuable discussions
throughout this work and his assistance in the preparation <strong>of</strong> this manuscript. This
paper was prepared from a portion <strong>of</strong> Ph.D. thesis submitted (by S. V.G.) to the Department
<strong>of</strong> Zoology, School <strong>of</strong> Graduate Studies, University <strong>of</strong> Toronto. This investigation was
supported by grants from the Province <strong>of</strong> Ontario (to S. V. G.) and National Research Council
<strong>of</strong> Canada (to R.A.L.).
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